The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases and drugs that target them. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are prototypes for family B polymerases that include human replicative DNA polymerases, but herpesvirus DNA polymerases are also excellent targets for antiviral drugs. This latter property is especially health-related, as we do not fully understand how the leading drug against herpes simplex virus (HSV) selectively inhibits the polymerase, and as new drugs are needed for treatment of herpesvirus infections, particularly infections resistant to current drugs. In this application, unanswered questions regarding the polymerase subunits, and drugs that target these proteins and their interaction are addressed. Specific aim 1 is to solve the structures of the (HSV DNA polymerase holoenzyme and/or this enzyme plus the other four viral replication fork proteins bound to primer-template. The structure of the holoenzyme bound to primer-template terminated with a dideoxynucleoside plus a deoxynucleoside triphosphate (dNTP) will be determined by X-ray crystallography, cryo-electron microscopy (cryo-EM) or both. Subsequent structures will include ones with acyclovir at the primer-terminus or acyclovir triphosphate as a mimic of the dNTP. Structures of replication fork complexes will be determined by cryo-EM. Specific aim 2 is to investigate the roles of two structural domains of Pol, pre-NH2 and NH2, whose biochemical functions are unknown. The hypothesis that a sequence motif of the pre-NH2 domain, which is conserved among herpesviruses and is important for viral DNA synthesis, interacts with another protein will be investigated using mass spectrometry of proteins bound to Pol from cells infected with viruses that are wild type or mutant for the motif and labeled with stable isotopes. Protein candidates identified will be investigated for their interaction with the motif and their roles during virus infection. A structural motif of the NH2 terminal domain that resembles RNA-binding proteins and is conserved among family B DNA polymerases will be investigated for its role in virus infection by construction and analyses of mutant viruses. Its biochemical function will be tested in assays testing the effects of mutations on Pol enzymatic activities and the strand displacement of RNA primers. Specific aim 3 is to discover new compounds that inhibit the interaction of HCMV Pol and UL44, and HCMV replication. The starting point is the structure of UL44 bound to a small covalent allosteric inhibitor of this interaction. Structure-guided medicinal chemistry will be used to increase the potency of this compound, and to improve its selectivity, to generate either new covalent or non-covalent inhibitors. Compounds will be tested for selective inhibition of the interaction, selective anti-HCMV activity in cells, and mechanisms of action and resistance.